Maintaining effective protein homeostasis, or proteostasis, is critical to the survival of all cells, and it protects against disease. Misfolded or unstructured proteins are functionally impaired; they have the potential to cause formation of potentially toxic aggregates. Critical components in cellular proteostasis networks are the mammalian stress-inducible heat shock protein-70 (HSP70, also called HSPA1A, HSP72) and its evolutionarily conserved bacterial ortholog, DnaK. These proteins coordinate key processes needed to maintain protein quality control, especially under conditions of enhanced stress; their activities include protein folding, protein transport across membranes, formation/dissolution of protein-protein interactions, and preventing accumulation of toxic protein aggregates. Thus, these molecular chaperones protect against the deleterious consequences of elevated physiologic and environmental stresses that cause conformational changes in polypeptides and disrupt protein function.
Altered protein quality control is characteristic of many human diseases. Cancer cells, for example, are subject to an enhanced stress environment that promotes protein misfolding; thus, these aberrant cells are especially dependent on the activities of HSP70 to maintain proteostasis. Indeed, HSP70 is considered a cancer-critical survival protein, and it is constitutively expressed at elevated levels in most cancers. This contrasts with its typically modest expression in unstressed normal cells; this has led to the testable suggestion that HSP70 inhibitors should exhibit a therapeutic index that can be exploited for preferential killing of cancer cells. Elevated HSP70 expression confers protection against a variety of stresses, such as hypoxia, altered metabolism and exposure to chemotherapeutic agents. It also inhibits apoptosis and oncogene-induced senescence, and is a major contributor to poor therapeutic outcome in patients. Reducing HSP70 abundance in cancer cells using RNA interference, antisense approaches, or peptides promotes apoptosis and cell cycle arrest, and increases sensitization to chemotherapeutic agents. In mammalian cells, the actions of HSP70 also coordinate with autophagy and proteasome pathways to aid in the disposal and recycling of altered proteins and organelles, and to prevent the accumulation of misfolded, or otherwise unwanted, proteins. Enhanced levels of autophagy are induced by different forms of stress, and promote the survival of established cancers. An impairment of HSP70 would be predicted to disrupt critical pathways of autophagy and the proteasome, particularly in tumor cells. HSP70 is a multifunctional HSP70 protein and a critical co-chaperone for the molecular chaperone HSP90. Working together, these chaperones direct the stability, localization and activity of a large and varied group of client proteins, many of which have been implicated in promoting tumorigenesis. A number of these client proteins have been implicated in the development and/or progression of cellular transformation; they include proteins involved in signal transduction, cell proliferation, or energy production. Examples of these proteins include AKT, BRAF(V600E) and EGFR, among others.
While HSP90 inhibitors are known, one of the consequences of HSP90 inhibition is a robust upregulation of HSP70, likely reducing the overall anti-tumor efficacy of HSP90 inhibitors. Because HSP70 is an obligate co-chaperone for HSP90, but also has independent pro-survival functions in tumor cells, targeting HSP70 has the potential to simultaneously impair multiple signaling- and protein quality control-networks. This strategy offers unique advantages for developing an effective and durable therapy for cancer treatment, even in genetically heterogeneous tumor cells.
Protein misfolding and aggregation contribute to cellular toxicity in disorders such as cystic fibrosis and certain neurodegenerative diseases, and increases in HSP70 expression can modulate this phenotype. Thus, HSP70 and DnaK have emerged as attractive targets for the development of new treatments for cancers and other human disorders, as well as to address the growing need for alternative antimicrobial agents. However, despite a great deal of interest in the translational potential of these chaperones, remarkably few selective, effective modulators have been identified.
HSP70 and DnaK are part of an evolutionarily conserved family of 70 kDa heat shock proteins that have a similar overall structure: they consist of an approximately 44 kDa N-terminal nucleotide binding domain (NBD), a conserved flexible middle linker, and the approximately 25 kDa C-terminal substrate/peptide binding domain (SBD). Each major domain is composed of several highly dynamic subdomains. HSP70 and DnaK exhibit about 45-50% amino acid similarity. These molecular chaperones transiently and dynamically interact with a very large and diverse array of substrates, or clients, by binding exposed hydrophobic regions of partially folded or unfolded proteins. When ADP is bound to the NBD, the substrate binds with high affinity; ATP binding favors substrate release. Allosteric coupling involves cycles of nucleotide binding and hydrolysis, together with the binding and release of substrate.
Many previous chemical screens for HSP70 inhibitors have tended to focus on the N-terminal ATP-binding site (or surrounding regions), partly because this approach was successful in identifying inhibitors of the HSP90 molecular chaperone as well as many protein kinases. However, the ATP-binding site of HSP70 has been less amenable to this strategy. While there is great interest in targeting HSP70 and DnaK for therapeutic use, there are very few selective or well-characterized compounds that directly interact with these proteins to modulate function. Further, there are no drugs in clinical trials that directly bind HSP70. 2-Phenylethynesulfonamide (referred to as PES, FIG. 1A), a biotinylated PES analog (referred to as B-PES, FIG. 1B) and a chlorinated derivative, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl, FIG. 1C) were previously identified as selective inhibitors of HSP70/DnaK. See, e.g., International Patent Application Publication No. WO2010/033771, published Mar. 25, 2010 and US Patent Application Publication No. US2011/0189125.